Cloning and analysis of PCR-generated DNA fragments. | Zendy

G L Costa | Zendy; Albert Grafsky | Zendy; Michael P. Weiner | Zendy

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Cloning and analysis of PCR-generated DNA fragments.

Author(s) -

G L Costa,

Albert Grafsky,

Michael P. Weiner

Publication year - 1994

Publication title -

genome research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.556

H-Index - 297

eISSN - 1549-5469

pISSN - 1088-9051

DOI - 10.1101/gr.3.6.338

Subject(s) - biology , cloning (programming) , primer (cosmetics) , microbiology and biotechnology , polymerase chain reaction , dna polymerase , primer dimer , taq polymerase , polymerase , dna , genetics , gene , thermus aquaticus , multiplex polymerase chain reaction , chemistry , computer science , organic chemistry , programming language

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.

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